Nonviral gene therapies hold great promise for the treatment of genetic and acquired diseases. However, increasing the efficiency of delivery and gene expression while reducing the inflammatory response remains a challenge. A recent study provided evidence that increased gene expression and decreased immune response result from sequentially injecting lipid and then DNA into mice, as opposed to delivering them simultaneously as a complex. It is hypothesized that the complexes formed when lipid and DNA are injected sequentially differ in structure and/or composition of bound serum proteins, leading to the variations observed in vector efficiency and toxicity. The goal of this research is to isolate the respective complexes and study their physical characteristics (i.e., size, structural details) by electron microscopy. Proteins unique to the various complexes will be examined by 2-D gel electrophoresis and the nature and identity of the proteins will be determined using mass spectroscopy. Similar studies will be performed in cationic polymer based nonviral vectors. These studies should provide useful information towards the development of new nonviral vectors with high gene expression and low immune stimulation.